THE POSSIBLE ROLE OF RECEPTOR CHAPERONE MOLECULE/INTERFERON- STIMULATED GENE RTP4 IN THE DEVELOPMENT OF ANTINOCICEPTIVE TOLERANCE TO MORPHINE

Abstract Background Although opioid analgesics exhibit potent antinociceptive effects, various side effects including antinociceptive tolerance limit their effective clinical use. Our previous studies show that neuronal receptor transporter protein 4 (RTP4), one of the receptor chaperone proteins that is also known as interferon (IFN)-stimulated gene, contributes to the mechanism of development of morphine tolerance. That is, the brain region (hypothalamus)-specific knock-down of RTP4 using AAV Cre recombinase in RTP4flox mice partially but significantly suppresses/delays the development of morphine tolerance (Fujita et al., 2022). Since chronic morphine treatment has been shown to increase Rtp4 in hypothalamus, this indicates a role for this chaperone in the mechanism of morphine tolerance. Aims & Objectives Although, studies suggest that glial as well as neuronal cells contribute to antinociceptive tolerance, the role of RTP4 in the specific cell types on opiate induced tolerance has not yet been elucidated. Here, we examined the changes in RTP4 levels in both neuronal and microglial cells after morphine exposure. Methods A neuronal cell line, Neuro2AMOPr cells, that express mu opioid recepor (MOPr) and a microglial cell line, SIM-A9 cells, were treated with DAMGO or morphine (10 µM to 1 mM), both are the MOPr agonist, for 24 hrs. The cells were collected and the RTP4 mRNA levels were evaluated by using RT-qPCR. Inhibitors were pretreated 30 to 60 min before the morphine treatment. Results We find that DAMGO treatment (10 µM, 24 hr) significantly up-regulates RTP4 mRNA levels in Neuro2AMOPr cell. This up-regulation was completely reversed by naltrexone, a MOPr antagonist and by U0126, a mitogen-activated protein kinase (MAPK) inhibitor. On the other hand, in SIM-A9 cells, only higher concentration of morphine (1 mM, 24-hr) significantly up-regulates RTP4 mRNA levels. This up- regulation was not reversed by naltrexone while it was significantly inhibited by a neutralizing antibody targeting toll-like receptor 4 (TLR4), antibody targeting IFN receptor, and by a janus kinase (JAK) inhibitor. Furthermore, interferon (IFN)-β mRNA levels were increased by morphine treatment in microglial cell. Discussion & Conclusion These findings suggest that in neuronal cells Rtp4 induction involves MOPr, Gi-mediated signaling and MAPK activation. Alternatively, Rtp4 induction in glial cells occurs via Toll-like receptor 4 and not MOPr. Together, these studies highlight a distinctive role for morphine activity in regulating RTP4 in neuronal and glial cells, which, in turn, may play an important role in modulating morphine effects in vivo. References Fujita, W. et al. (2022) 'Receptor transporter protein 4 (RTP4) in the hypothalamus is involved in the development of antinociceptive tolerance to morphine,' Biomolecules, 12(10), p. 1471. https://doi.org/10.3390/biom12101471.